The present invention relates generally to methods for purifying proteins from biological media and, more particularly, to methods for purifying proteins containing EGF-like domains from conditioned medium.
The recombinant production of high value proteins has become widespread. Typically, a prokaryotic or eukaryotic cellular source is transformed with recombinant DNA encoding the protein of interest, and the transformed cells are grown in a suitable culture medium. The proteins may then be recovered from the growing cells, either by harvesting the cells and disrupting them to release the proteins or by collecting conditioned medium into which the recombinant protein has been secreted. In either case, it is necessary to recover and purify the recombinant protein from the cell culture.
Protein purification may be achieved by a variety of processes, such as chromatography including ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reversed-phase liquid chromatography, and the like. Usually, several separation steps will be performed sequentially in order to recover the recombinant protein at the desired level of purity.
In each step of a protein purification process, competing objectives of purity and recovery must be satisfied. Generally, the higher the purity achieved, the lower the recovery. Because of the high value of most recombinant protein products, it is of great interest to identify separation methods and steps which provide for enhanced purity and recovery with particular proteins or groups of proteins. In addition, it is desirable that the separation methods contain relatively few separation steps and that the chromatographic materials used in separation be easily cleaned and reused. Additionally, it is desirable to employ the least costly chromatographic materials available for performing any particular purification method.
Thrombomodulin is a 105 kD cell membrane protein which possesses anticoagulant activity. Recombinant thrombomodulin (particularly soluble thrombomodulin analogs) promises to be therapeutically useful in the treatment of blood clots and other thrombotic circulatory conditions, such as coronary and pulmonary embolism, strokes, myocardial infarction, disseminated intravascular coagulation, deep vein thrombosis, septic shock, and the prevention of reocclusion following thrombolytic therapy.
Heretofore, purification of soluble recombinant thrombomodulin has been achieved by passage of a conditioned medium over a strong anionic column, such as a quaternary aminoethyl (Q-) Sepharose.RTM. column, followed by affinity chromatography using a thrombin column. While acceptable purity can be achieved, the need to employ an affinity column is expensive and provides a possible source of contamination (in the event that the thrombin is lost from the column during the purification procedure).
The use of affinity chromatography is necessitated by the presence of bovine serum albumin (BSA) in conditioned medium which has been supplemented with calf serum. BSA has an isoelectric point very close to that of thrombomodulin, and separation efficiency suffers during the anion exchange step.
It would therefore be desirable to provide improved methods for the separation and purification of thrombomodulin and similar recombinantly produced proteins from conditioned medium. It would be particularly desirable if the methods provided for very high levels of purification while minimizing losses of the valuable recombinant protein. It would be further desirable if the methods were suitable for purifying very large quantities of the proteins, while utilizing relatively inexpensive chromatographic materials and extending the useful life of such materials.